In basic research on the hepatitis C virus (hereinafter, also referred to as HCV) and research and development of anti-HCV drugs, an experimental system that enables efficient virus amplification is essential. Specifically, a system for amplifying HCV in cultured cells and a system for evaluating the propagation of HCV in cultured cells are necessary, and it is considered that construction of such systems will allow dramatic progress in the research mentioned above to be realized.
HCV is a virus belonging to the family Flavivirus. It comprises a single-stranded (+) sense RNA as its genome, and it is known to cause hepatitis C. HCV is classified into many types depending on genotype or serotype. According to phylogenetic analysis conducted by Simmonds et al. using nucleotide sequences of HCV strains, HCV is classified into genotypes 1 to 6, and each type is further classified into several subtypes (Simmonds et al., Hepatology, 1994, Vol. 10, pp. 1321-1324). The full length genome nucleotide sequences of a plurality of HCV genotypes have been determined (Choo et al., Science, 1989, Vol. 244, pp. 359-362, Kato et al., Journal of Medical Virology, 1992, Vol. 64, pp. 334-339, Okamoto et al., Journal of General Virology, 1992, Vol. 73, pp. 673-679 and Yoshioka et al., Hepatology, 1992, Vol. 16, pp. 293-299).
HCV infection is spreading all over the world. In Japan, the U.S.A., and Europe, the proportion of patients infected with HCV of genotype 1 is high. In contrast, the proportion of patients infected with HCV of genotype 3 is high in India, Nepal, Pakistan, and Australia (Gravitz, Nature, 2011, Vol. 474, pp. s2-s4 and Rehman et al., Genetic Vaccines and Therapy, 2011, Vol. 9, pp. 2-5).
Until recently, infection of cultured cells with HCV and replication of HCV genomes in cultured cells have been impossible. Accordingly, studies on mechanisms of HCV replication and infection have required in vivo experiments using chimpanzees as experimental animals. However, subgenomic replicon RNAs have been produced from the Con1 strain, the HCV-N strain, the HCV-O strain belonging to HCV genotype 1b, and the H77c strain belonging to HCV genotype 1a. This has enabled studies on the HCV replication mechanism via in vitro experiments using cultured cells (JP 2001-17187 A and Lohmann et al., Science, 1999, Vol. 285, pp. 110-113, Blight et al., Science, 2000, Vol. 290, pp. 1972-1974, Friebe et al., Journal of Virology, 2001, Vol. 75, pp. 12047-12057 and Ikeda et al., Journal of Virology, 2002, Vol. 76, pp. 2997-3006). Herein, the subgenomic replicon RNA of HCV means an RNA which comprises a portion of HCV genome, and can autonomously replicate an RNA derived from the HCV genome when introduced into cultured cells, but does not have an ability to produce infectious HCV particles.
In addition to subgenomic replicon RNAs, full-genomic replicon RNAs producing infectious HCV particles in vitro have been produced from the JFH-1 strain belonging to HCV genotype 2a. This has enabled studies on the HCV infection mechanism via in vitro experiments using cultured cells (Kato et al., Gastroenterology, 2003, Vol. 125, pp. 1808-1817 and Wakita et al., Nature Medicine, 2005, Vol. 11, pp. 791-796). Herein, the full-genomic replicon RNA of HCV means an RNA which comprises the full-length HCV genome; i.e., a 5′ untranslated region, structural genes, non-structural genes, and a 3′ untranslated region, and can autonomously replicate an RNA derived from the HCV genome when introduced into cultured cells.
At present, RNAs that can produce infectious HCV particles in an in vitro system using cultured cells are limited to those derived from the JFH-1 strain of genotype 2a. RNAs capable of mass-producing HCV particles in an in vitro system for obtaining raw material of an HCV vaccine are limited to HCV of the JFH-1 strain or a full-genomic replicon derived from the JFH-1 strain.
The main therapeutics for hepatitis C are monotherapy using interferon-α or interferon-β and combined therapy using interferon-αα and ribavirin, which is a purine nucleoside derivative. Such therapy, however, is recognized as having a therapeutic effect in only about 60% of all subjects, and it is known that hepatitis C recurs in more than half of even those patients for whom the therapy was effective, in cases in which the therapy was stopped. The therapeutic effect of interferon is associated with HCV genotype, and it is known that the effect on genotype 1b is low and that the effect on genotype 2a or 3a is higher (Mori et al., Biochemical and Biophysical Research Communications, 1992, Vol. 183, pp. 334-342). While the causes of differences in interferon therapeutic effects depending on HCV genotype remain unknown, differences in HCV replication mechanism or replication efficiency are considered to be among the causes.
In recent years, novel therapeutic agents against hepatitis C such as inhibitors against HCV-derived protease or polymerase, have been developed. However, it is reported that TMC435, which is an HCV NS3/4A protease inhibitor, has strong inhibitory effects on genotypes 1 to 6 except for genotype 3a, but weak inhibitory effects on genotype 3a; that is, inhibitory effects against HCV vary depending on genotype (Reesink et al., Gastroenterology, 2010, Vol. 138, pp. 913-921).
The HCV subgenomic replicon RNAs that have been produced are, however, limited to several types derived from HCV strains of genotypes 1a, 1b, and 2a. Full-genomic replicon RNAs capable of producing infectious HCV particles that have been produced are limited to those derived from the genome of the JFH-1 strain of genotype 2a or those derived from a chimeric genome composed of structural genes derived from a strain other than the JFH-1 strain and non-structural genes of the JFH-1 strain. It is therefore difficult to elucidate the correlation between HCV genotype and HCV replication mechanism or replication efficiency. At present, unfortunately, HCV particles that can be artificially prepared as raw materials for HCV vaccines are limited to those of genotype 2a.
In studies using subgenomic replicon RNAs or full-genomic replicon RNAs derived from HCV of the same genotype, HCV replication mechanisms or replication efficiencies cannot be compared between different genotypes. Accordingly, no clues regarding the development of anti-HCV drugs that exert therapeutic effects independently of genotype have been found.
In research and medical fields related to HCV, specifically, obtaining an HCV strain of genotype 3a and production of replicon RNA thereof are strongly demanded in developing genotype-independent anti-HCV drugs and, in particular, anti-HCV drugs against HCV of genotype 3a.
Accordingly, it could be helpful to provide a novel HCV strain of genotype 3a, and, further, replicon RNA having autonomous replication ability derived from a novel HCV strain of genotype 3a.